CN109260174B - High-throughput preparation method of therapeutic protein nanoparticles - Google Patents
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- C07K1/30—Extraction; Separation; Purification by precipitation
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/28—Insulins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/38—Albumins
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/38—Albumins
- A61K38/385—Serum albumin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- A—HUMAN NECESSITIES
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
Abstract
The invention discloses a high-flux preparation method of therapeutic protein nanoparticles. The method comprises the following steps: firstly, converting hydrophilic protein drugs into protein-lipid complexes by using a hydrophobic ion pair method, then diluting the protein-lipid complexes by using an organic solvent which is mutually soluble with water, and then preparing the protein nanoparticles by using a rapid nano-precipitation method. Compared with the traditional preparation method of the protein nanoparticles, the method has the greatest advantages that the method can be used for continuous large-scale production, the productivity can reach 1.2 g/h, and the production capacity is far higher than that of the traditional preparation method of the protein nanoparticles. In addition, the rapid nano-precipitation method can be used for continuous production, has small difference among batches, and is suitable for industrial production. Further, the hyaluronic acid coated on the surface of the protein particles prepared by the present invention can impart stability for long-term storage and mucus penetration to the protein drug nanoparticles. The protein nano-particles prepared by the method have a wide application prospect in the aspect of in vivo delivery of protein medicaments.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a high-throughput preparation method of protein nanoparticles.
Background
The biotechnology drugs mainly include polypeptide drugs, protein drugs and nucleic acid drugs. However, most of the biological drugs are protein polypeptides, and among the biotechnological drugs that have been marketed and developed, the protein drugs account for the vast majority, mainly including cytokine drugs and recombinant hormone drugs. At present, protein polypeptide drugs have irreplaceable effects in diseases such as tumors, autoimmune deficiency diseases, cardiovascular diseases and the like because of high specificity and excellent curative effect.
However, compared with conventional drugs, protein polypeptide drugs have a short half-life in vivo due to their large molecular weight, poor stability, variability, and difficulty in penetrating intestinal mucosa, thus limiting their wide application. In recent years, carriers such as liposome, micelle and hydrogel are developed for in vivo delivery of protein polypeptide drugs, but these delivery systems are often limited by their low drug encapsulation efficiency and complex preparation processes, and are difficult to scale up and convert clinically.
Rapid nano-precipitation is a microfluidic technique that can prepare drug nanoparticles by kinetically controlled molecular aggregation processes. And the method can be used for continuous large-scale production and is suitable for industrial production. The main mechanism is to realize the fast exchange between solvent (containing medicine) and non-solvent (containing stabilizer) by means of high-turbulence mixer (such as coaxial turbulence mixer, four-channel vortex mixer, etc.), and to control the grain size and dispersivity of nanometer grains by regulating the nucleation and growth rate of solute. And drug nanoparticles have been mass-produced using this method. However, one limitation of the rapid nano-precipitation method is that it is only suitable for hydrophobic small molecule drugs, but not for biotechnological drugs such as protein polypeptides.
Disclosure of Invention
The invention aims to provide a high-throughput preparation method of protein nanoparticles aiming at the defects and shortcomings in the prior art. According to the method, by combining a hydrophobic ion pair technology and a rapid nano-precipitation method, a hydrophilic protein drug is converted into an oil-soluble protein-lipid complex by using the hydrophobic ion pair method, and then the protein nano-particles are prepared by using the rapid nano-precipitation method.
It is another object of the present invention to provide a lyophilized formulation of therapeutic protein nanoparticles.
The above object of the present invention is achieved by the following scheme:
a high-flux process for preparing the protein nanoparticles of therapeutic nature includes such steps as preparing the protein-lipid composition from hydrophilic protein medicine by hydrophobic ion pair method, diluting the protein-lipid composition by water-soluble organic solvent, and fast nano-deposition.
Preferably, the process of the hydrophobic ion pair method comprises: mixing a protein aqueous solution and a dichloromethane or trichloromethane solution of lipid with opposite charges to the protein in an equal volume, wherein the mass ratio of the protein to the positive charged lipid is 1: 1-8, adding an organic solvent which is mutually soluble with water to form a homogeneous solution, adding water with an equal volume to the protein aqueous solution, centrifuging and layering to obtain a lower organic phase which is a solution dissolved with the protein-lipid complex. In preparing the aqueous protein solution, in order to facilitate the dissolution of the protein, the protein may be dissolved using a dilute acid solution, and then the solution may be neutralized with an alkali.
Preferably, the mass ratio of the protein to the lipid is 1: 4-8 after the protein aqueous solution and the lipid solution are mixed. When the mass ratio of the protein to the lipid is within the proportion range, the transfer rate of the protein drug from the water phase to the organic phase is very high and exceeds 97 percent during extraction, namely the utilization rate of the protein drug is higher in the preparation process.
Preferably, the water-miscible organic solvent is ethanol.
Preferably, the process of the rapid nanoprecipitation method comprises: diluting the protein-lipid complex solution prepared by the hydrophobic ion pairing method with ethanol according to the volume ratio of 1: 5-11, introducing the diluted protein-lipid complex solution and a phosphate buffer solution with the concentration of 0.5-10 mM into different channels in a vortex mixer according to the volume ratio of 1: 3-9, wherein the flow rate of an organic phase is 1 mL/min-10 mL/min, and mixing through high-speed turbulence to prepare the protein nanoparticle solution.
In the rapid nano-precipitation process, the dilution ratio of the protein-lipid complex, the concentration of the solute in the phosphate buffer solution, and the ratio of the ethanol solution of the protein-lipid complex to the phosphate buffer solution all affect the particle size of the protein nanoparticles, and only when the 3 conditions or parameters are within the above range, the protein nanoparticles with proper particle size and high uniformity of particle size can be prepared.
Preferably, the concentration of solute in the phosphate buffer solution is 0.5-1 mM; in the vortex mixer, the volume ratio of the organic solution to the aqueous phase was 1:7, respectively, and the flow rate of the organic phase was 5 mL/min. Under the condition, the prepared protein nanoparticles have the best repeatability and uniformity, the particle size is better, and the average particle size is 35 nm.
Preferably, the protein nanoparticle solution and the negative electrode polymer aqueous solution are respectively introduced into different channels of a vortex mixer, so that the protein nanoparticles stably coated with the negative electrode polymer can be prepared; wherein the flow rate of the protein nanoparticle solution is 1 mL/min-40 mL/min, and the flow rate of the negative electric polymer is 0.1 mg/mL-0.5 mg/mL.
Preferably, the protein drug is insulin, ovalbumin or human serum albumin.
More preferably, the protein drug is insulin.
Preferably, the lipid is DDAB or DOTAP.
Preferably, the negatively charged polymer includes, but is not limited to, hyaluronic acid, sodium alginate or polyglutamic acid.
The invention also discloses a freeze-dried preparation containing the protein nanoparticles prepared by the method.
Preferably, a freeze-drying protective agent is added into the protein nanoparticle solution, and the freeze-dried preparation can be obtained through freezing and drying.
Preferably, the lyoprotectant is one or more of mannitol, xylitol, glycine or sorbitol.
More preferably, the lyoprotectant is a mixture of mannitol and xylitol.
Preferably, the ratio of the mass of the mannitol to the mass of the xylitol to the volume of the protein nanoparticle solution is 0-10 g:100 mL.
More preferably, the ratio of the mass of mannitol to the mass of xylitol to the volume of the protein nanoparticle solution is 0.5g: 1g:100 mL.
Compared with the prior art, the invention has the following beneficial effects:
compared with the traditional preparation method of the protein nanoparticles, the method has the greatest advantages that the method can be used for continuous large-scale production, the productivity can reach 1.2 g/h, and the production capacity is far higher than that of the traditional preparation method of the protein nanoparticles. In addition, the rapid nano-precipitation method can be used for continuous production, has small difference among batches, and is suitable for industrial production.
Further, the hyaluronic acid coated on the surface of the protein particles prepared by the present invention can impart stability for long-term storage and mucus penetration to the protein drug nanoparticles. The protein nano-particles prepared by the method have a wide application prospect in the aspect of in vivo delivery of protein medicaments.
Drawings
FIG. 1 is a graph showing the effect of the ratio between insulin and DDAB on the phase transfer rate of insulin.
FIG. 2 (a) particle size plot of free insulin in water and insulin-DDAB in organic solvent, (b) circular dichroism spectra of free insulin and insulin-DDAB hydrophobic ion pair complexes.
FIG. 3 is a graph showing the effect of phosphate buffer salt concentration on the particle size of insulin nanoparticles.
Figure 4 is the batch-to-batch variability of insulin nanoparticle preparation.
FIG. 5 is a graph showing the effect of flow rate on HA coating on the surface of insulin nanoparticles.
FIG. 6 shows the in vivo hypoglycemic effect of the lyophilized formulation of insulin nanoparticles.
Detailed Description
The present invention is further described in detail below with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1
Taking insulin as an example of a protein drug, the method for preparing the insulin nano-particles comprises the following steps:
(1) hydrophobic ion pairing process
Weighing insulin (insulin) and dissolving the insulin in 0.01M dilute hydrochloric acid solution to prepare 5mg/mL insulin solution, and adjusting the pH of the solution to 7.4 by using NaOH for later use;
weighing DDAB, dissolving in dichloromethane to prepare a series of DDAB organic solutions (the mass concentration is 5 mg/mL-40 mg/mL);
③ mixing the insulin solution and the DDAB dichloromethane solution (both 5 mL) with the same volume, adding ethanol with double volume (10 mL) to form a single-phase solution, slightly shaking at 100 rpm for 30 minutes, adding 5mL pure water to re-phase, and centrifuging to obtain a lower organic phase which is the solution dissolved with the insulin-lipid (insulin-DDAB) compound.
The phase transfer rate of insulin from the aqueous phase to the organic phase was calculated by measuring the amount of insulin in the supernatant.
The measurement results are shown in FIG. 1, and it is understood from the figure that when the mass ratio of DDAB to insulin is increased from 1:1 to 4: 1, the phase transfer rate is gradually increased from 38.8% to 97.2%, the mass ratio of DDAB to insulin is continuously increased, and the phase transfer rate is gradually close to 100%.
The results of the dynamic light scattering test of the solutions of the insulin aqueous solution and the insulin-lipid complex are shown in fig. 2. As can be seen from FIG. 2, the particle sizes of the insulin-DDAB complex in the free insulin aqueous solution and the organic solvent were 2.4 and 3.2 nm, respectively (FIG. 2 a), demonstrating that the insulin-DDAB complex was still present in the solvent as a single molecule. Furthermore, as can be seen from FIG. 2b, the results of the circular dichroism spectroscopy of the free insulin and insulin-DDAB complex in the aqueous insulin solution are almost identical, thus demonstrating that the conformation and function of insulin in the insulin-DDAB complex are not changed.
(2) Process of nano precipitation method
The dichloromethane solution of the insulin-lipid complex prepared by the method is diluted by ethanol (volume ratio is 1: 9), and is introduced into the 1 st channel of a four-channel vortex mixer, and phosphate buffer solution is introduced into the other three channels, so that rapid turbulent mixing is realized to prepare the insulin nanoparticle solution. The volume ratio of the organic solution to the water phase is 1: 3-9, and the flow rate of the organic phase is 1-10 mL/min.
When the salt concentration of the phosphate buffer is changed, insulin nanoparticles having different particle sizes can be prepared, and the specific relationship between the salt concentration and the particle size is shown in fig. 3.
When the salt concentration of the phosphate buffer solution is 0.5-10 mM, the particle size range of the prepared insulin nanoparticles is 35-792 nm, and the dispersity is 0.1-0.2, which shows that the particle size distribution of the insulin nanoparticles in the obtained insulin nanoparticle solution is uniform.
When the volume ratio of the organic solution to the aqueous phase is 1:7, the flow rate of the organic phase is 5mL/min, and the salt concentration of the phosphate buffer is 0.5 mM, the preparation of the insulin nanoparticles is repeated three times under the condition, the particle size results are shown in FIG. 4, and the average particle size is 35nm, which proves the good repeatability of the method.
(3) Hyaluronic acid is coated on surfaces of insulin nanoparticles
Taking Hyaluronic Acid (HA) as an example, the coating is coated on the surface of the insulin nanoparticle as a protective layer, and the specific process is as follows:
and (3) dissolving a hyaluronic acid solution in water to prepare a 0.2 mg/mL aqueous solution, introducing the insulin nanoparticle solution obtained in the step (2) into a 1 st channel and a 2 nd channel in a four-channel vortex mixer, introducing an HA aqueous solution into a 3 rd channel and a 4 th channel, wherein the flow rates of the four channels are consistent, adjusting the flow rates of the channels to be 1mL/min, 2 mL/min, 5mL/min, 10mL/min, 20mL/min and 40mL/min respectively, and enabling the two liquids to reach a vortex mixing area through the four channels to be mixed to obtain the insulin nanoparticles with the HA coated surfaces.
The prepared HA-coated insulin nanoparticles have different particle sizes when the flow rates of the channels are different, and the specific result is shown in FIG. 5, wherein NP refers to protein nanoparticles which are not coated with hyaluronic acid. As can be seen from the figure, when the flow rate is less than 20mL/min, the particle size of the particles increases with the decrease of the flow rate, and when the flow rate is between 20 and 40mL/min, the particle size of the particles does not change much. When the flow rate is 20mL/min, the obtained particles are most uniform, and the potential of the particles is changed from +10 mV to-20 mV. The preferred flow rate of 20mL/min was chosen.
(4) Preparation of HA-surface-coated insulin nanoparticle lyophilized preparation
The preparation process of the freeze-dried preparation comprises the following steps: adding mannitol, xylitol, glycine, sorbitol or their composition into insulin nanoparticle water solution coated with HA on surface, stirring, mixing, freezing with liquid nitrogen for 10 min, and drying at-30 deg.C under 0.37bar vacuum for 48 hr to obtain lyophilized nanometer preparation.
Through experimental screening, the optimal freeze-drying protective agent of the insulin nanoparticle aqueous solution with the HA coated on the surface is a mannitol/xylitol composition. Wherein the ratio of the mass of mannitol to the mass of xylitol to the volume of the drug-loaded nanoparticle aqueous solution is 0-10 g:100 mL; the optimal ratio is 0.5g to 1g to 100 mL.
The results of comparing the properties of the HA-surface-coated insulin nanoparticles before and after lyophilization are shown in table 1.
Table 1 results of various properties of HA-surface-coated insulin nanoparticles before and after lyophilization
As can be seen from table 1, the properties of the HA-coated insulin nanoparticles after lyophilization were not significantly changed compared to the drug-loaded nanoparticles before lyophilization, and the reconstitution time was very short, and the drug-loaded nanoparticles were completely reconstituted within 5 seconds. The composite freeze-drying protective agent is proved to be capable of well protecting the nano particles in the freeze-drying process.
Example 2 hypoglycemic effect of HA-surface coated insulin nanoparticle lyophilized formulation prepared in example 1
After the insulin nanoparticles coated with HA on the surface prepared in example 1 are dialyzed, a freeze-drying protective agent is added for freeze-drying to obtain a powder preparation, and the powder preparation is filled into gelatin enteric capsules to obtain the samples to be tested.
The testing process comprises the following steps: injecting 75 mg/kg streptozotocin into the abdominal cavity of 180-220 g SD rats to induce the SD rats to become type I diabetes model rats, and dividing the SD rats into 4 groups after the blood sugar of the model rats is stabilized, wherein each group comprises 6 rats. Before the experiment, all experimental groups were fasted overnight, group 1 was gavaged with normal saline, group 2 was gavaged with a capsule containing insulin powder (48 IU/kg), group 3 was gavaged with a capsule containing freeze-dried powder of insulin nanoparticles coated with HA on the surface (75 IU/kg), and group 4 was injected with insulin solution subcutaneously (5 IU/kg). Blood was taken from the tip of the tail every 1 hour, and blood glucose was measured by a glucometer.
As shown in FIG. 6, it can be seen that the blood glucose of the mice injected with subcutaneous insulin rapidly drops to a low level, but the mice are easy to have a risk of hypoglycemia, and the time for lowering the blood glucose is short, and the blood glucose concentration begins to rise 2h after the injection. The good effect of the oral insulin nanoparticle preparation can be attributed to the insulin release mechanism of the insulin nanoparticle preparation. After the insulin nano-particles enter blood, with the dissociation or degradation of HA coated on the outer layer of the particles, because of the hydrophobicity of the insulin-DDAB hydrophobic ion pair compound, insulin can not burst in blood and is slowly released at a constant speed, so that the blood sugar is promoted to stably drop. The oral insulin powder capsule served as a negative control and had substantially no effect on blood glucose.
From the above examples, the high-throughput preparation method of protein nanoparticles provided by the invention can be used for continuous large-scale production, which is much higher than the production capacity of the traditional preparation method of protein particles; the difference between batches is small, continuous production is realized, and the method is suitable for industrial production; and the performance and the function of the prepared protein nanoparticles are not damaged.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (6)
1. A high-flux preparation method of therapeutic protein nanoparticles is characterized in that a hydrophobic ion pair method is utilized to convert hydrophilic protein drugs into protein-lipid complexes, and then the protein-lipid complexes are diluted by organic solvents mutually soluble with water and then prepared into the protein nanoparticles by a rapid nano-precipitation method;
the process of the hydrophobic ion pair method comprises the following steps: mixing a protein water solution and a methylene dichloride or trichloromethane solution of lipid with opposite charges of protein in an equal volume, wherein the mass ratio of the protein to the lipid is 1: 1-8, adding an organic solvent which is mutually soluble with water to form a homogeneous solution, adding water in an equal volume with the protein water solution, centrifuging and layering to obtain a lower organic phase which is a solution dissolved with a protein-lipid complex;
the process of the rapid nano-precipitation method comprises the following steps: diluting the protein-lipid complex solution prepared by the hydrophobic ion pairing method with ethanol according to the volume ratio of 1: 5-11, introducing the diluted protein-lipid complex solution and a phosphate buffer solution with the concentration of 0.5-10 mM into different channels in a vortex mixer according to the volume ratio of 1: 3-9, wherein the flow rate of an organic phase is 1 mL/min-10 mL/min, and mixing through high-speed turbulence to prepare a protein nanoparticle solution;
respectively introducing the protein nanoparticle solution and the negative electric polymer aqueous solution into different channels of a vortex mixer to prepare protein nanoparticles stably coated with the negative electric polymer; wherein the flow rate of the protein nanoparticle solution is 20mL/min to 40mL/min, and the flow rate of the negative electric polymer is 0.1mg/mL to 0.5 mg/mL;
the protein medicine is insulin, ovalbumin or human serum protein; the lipid is didodecyldimethylammonium bromide or (2, 3-dioleoyl-propyl) -trimethylammonium chloride; the negative charge polymer comprises hyaluronic acid, sodium alginate or polyglutamic acid.
2. The method for preparing the therapeutic protein nanoparticles according to claim 1, wherein the mass ratio of the protein to the lipid is 1: 4-8 after the aqueous solution of the protein and the lipid solution are mixed.
3. The method for high throughput preparation of therapeutic protein nanoparticles according to claim 1, wherein the concentration of the solute in the phosphate buffer is 0.5-1 mM; in the vortex mixer, the volume ratio of the organic solution to the aqueous phase was 1:7, respectively, and the flow rate of the organic phase was 5 mL/min.
4. A lyophilized formulation of therapeutic protein nanoparticles comprising protein nanoparticles prepared by the method of any one of claims 1 to 3.
5. The lyophilized therapeutic protein nanoparticle formulation according to claim 4, wherein a lyoprotectant is added to the protein nanoparticle solution, and the lyophilized formulation is obtained by freezing and drying.
6. The lyophilized formulation of therapeutic protein nanoparticles of claim 5, wherein the lyoprotectant is one or more of mannitol, xylitol, glycine, or sorbitol.
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